Dendritic cells (DC) have been identified as a pivotal antigen presenting cell for initiation of an immune responses. It has been postulated that dendritic cells provide the basis for more effective immune responses, particularly for antigens wherein conventional vaccination is inadequate, or for use in producing a response to tumor antigens.
“Antigen presentation” is the set of events whereby cells fragment antigens into peptides, and then present these peptides in association with products of the major histocompatibility complex, (MHC). The MHC is a region of highly polymorphic genes whose products are expressed on the surfaces of a variety of cells. T cells recognize foreign antigens bound to only one specific class I or class II MHC molecule. The patterns of antigen association with either a class I or class II MHC molecule determines which T cells are stimulated.
T cells do not effectively respond to antigen unless the antigen is processed and presented to them by the appropriate antigen presenting cells (APC). The two major classes of antigen presenting cells are dendritic cells (DC) and macrophages. DC precursors migrate from bone marrow and circulate in the blood to specific sites in the body where they mature. This trafficking is directed by expression of chemokine receptors and adhesion molecules. Immature dendritic cells (DC) reside in the periphery and act as sentinels, detecting invasion by pathogenic microorganisms (Caetano, Immunity 14, 495-498, 2001). Exposure to certain agents trigger DC to differentiate and migrate to primary lymphoid organs where they present antigen to T cells and initiate a protective immune response (Banchereau, et al., Ann. Rev. Immunol. 18, 767-811, 2000; Banchereau & Steinman, Nature 392, 245-252, 1998). Tissue resident DC include Langerhans cells in skin, hepatic DC in the portal triads, mucosal DC and lung DC. Upon exposure to antigen and activation signals, the tissue resident DC are activated, and leave tissues to migrate via the afferent lymphatics to the T cell rich paracortex of the draining lymph nodes. The activated DC then secrete chemokines and cytokines involved in T cell homing and activation, and present processed antigen to T cells. In summary, dendritic cell precursors migrate to the primary lymphoid organs where they differentiate into mature dendritic cells.
Mature DC have a distinct morphology characterized by the presence of numerous membrane processes. These processes can take the form of dendrites, pseudopods or veils. DC are also characterized by the cell surface expression of large amounts of class II MHC antigens and the absence of lineage markers, including CD14 (monocyte), CD3 (T cell), CD19, 20, 24 (B cell), CD56 (natural killer), and CD66b (granulocyte). DC express a variety of adhesion and co-stimulatory molecules, e.g. CD80 and CD86, and molecules that regulate co-stimulation, such as CD40. The phenotype of DC varies with the stage of maturation and activation, where expression of adhesion molecules, MHC antigens and co-stimulatory molecules increases with maturation. Antibodies that preferentially stain mature DC include anti-CD83 and CMRF-44.
Activated DC are uniquely capable of processing and presenting antigens to naive T cells. The efficacy of DC in antigen presentation is widely acknowledged, but the clinical use of these cells is hampered by the fact that there are very few in any given organ. Animal studies demonstrate that mature DC can be generated ex vivo, loaded with antigen, and infused in vivo to trigger protective responses against tumors and pathogenic microorganisms (Fields et al., Proc. Natl. Acad. Sci 95: 9482-9487, 1998; Okada, H. et al. Int. J. Cancer 78: 196-201, 1998; Su et al., J. Exp. Med. 188: 809-818, 1998; DeMatos et al., J. Surg. Oncol., 68: 79-91, 1998; Zhu et al., J. Med. Primatol 29: 182-192, 2000). Large numbers of mature DC are required for this type of immunotherapy. These are typically generated by incubating human peripheral blood monocytes with GM-CSF plus IL-4 for one week, followed by monocyte-conditioned medium for 2-7 days (Gluckman et al., Cytokines Cell Mol Ther 3: 187-196, 1997; Chapuis et al., Eur. J. Immunol. 27: 431-441, 1997; Palucka et al., J. Immunol 160: 4587-4595, 1999). In human blood, for example, about 1% of the white cells are DC. While DC can process foreign antigens into peptides that immunologically active T cells can recognize, the low numbers of DC makes their therapeutic use very difficult. Thus, this process is not only lengthy and complex, but does not uniformly generate DC with full functional activity due to difficulties in standardizing the monocyte-conditioned medium (Tarte et al., Leukemia 14, 2182-2192, 2000; Verdijk et al., J. Immunol. 163, 57-61, 1999; Syme & Gluck, J. Hematother. Stem Cell Res. 10: 43-51, 2001). Thus, a need remains to generate mature dendritic cells in vitro.